JP5097247B2 - Confocal microscope apparatus and observation method using confocal microscope apparatus - Google Patents

Confocal microscope apparatus and observation method using confocal microscope apparatus Download PDF

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JP5097247B2
JP5097247B2 JP2010158854A JP2010158854A JP5097247B2 JP 5097247 B2 JP5097247 B2 JP 5097247B2 JP 2010158854 A JP2010158854 A JP 2010158854A JP 2010158854 A JP2010158854 A JP 2010158854A JP 5097247 B2 JP5097247 B2 JP 5097247B2
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竜男 中田
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    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
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    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/0004Microscopes specially adapted for specific applications
    • G02B21/002Scanning microscopes
    • G02B21/0024Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052Optical details of the image generation
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/0004Microscopes specially adapted for specific applications
    • G02B21/002Scanning microscopes
    • G02B21/0024Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052Optical details of the image generation
    • G02B21/0072Optical details of the image generation details concerning resolution or correction, including general design of CSOM objectives
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/0004Microscopes specially adapted for specific applications
    • G02B21/002Scanning microscopes
    • G02B21/0024Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052Optical details of the image generation
    • G02B21/0076Optical details of the image generation arrangements using fluorescence or luminescence
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/0004Microscopes specially adapted for specific applications
    • G02B21/002Scanning microscopes
    • G02B21/0024Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/008Details of detection or image processing, including general computer control
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/16Microscopes adapted for ultra-violet illumination ; Fluorescence microscopes
    • GPHYSICS
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    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
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    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/32Micromanipulators structurally combined with microscopes

Description

本発明は、蛍光色素や蛍光タンパクで標識された試料を、励起波長を用いて励起して、前記試料から放出された蛍光を検出する共焦点顕微鏡装置及び共焦点顕微鏡装置を用いた観察方法に関する。   The present invention relates to a confocal microscope apparatus that excites a sample labeled with a fluorescent dye or a fluorescent protein using an excitation wavelength and detects fluorescence emitted from the sample, and an observation method using the confocal microscope apparatus .

標本の走査画像を得るための第1の走査光学系と、標本の特定の部位に特異現象を発現させるための第2の走査光学系とを備えた走査型レーザ顕微鏡が提案されている(特開2000−275529号公報参照)。この走査型レーザ顕微鏡では、第1の走査光学系におけるレーザ光源及び光路で標本面上の特定部位を照射して標本又は標本に注入された化学物質に刺激を加え、第2の走査光学系におけるレーザ光源及び光路で標本面上の上記とは異なる特定部位を励起して蛍光を検出し画像化する。なお、本明細書では、特に明記しない限り、標本の画像取得するための走査光学系を「第1の走査光学系」と称し、標本の特定の部位に特異現象を発現させるための走査光学系を「第2の走査光学系」と称する。   There has been proposed a scanning laser microscope including a first scanning optical system for obtaining a scanning image of a specimen and a second scanning optical system for causing a specific phenomenon to occur at a specific part of the specimen (particularly, No. 2000-275529). In this scanning laser microscope, a specific part on the specimen surface is irradiated with the laser light source and optical path in the first scanning optical system to stimulate the specimen or the chemical substance injected into the specimen, and in the second scanning optical system. Fluorescence is detected and imaged by exciting a specific part different from the above on the specimen surface with a laser light source and an optical path. In this specification, unless otherwise specified, a scanning optical system for acquiring an image of a specimen is referred to as a “first scanning optical system”, and a scanning optical system for causing a specific phenomenon in a specific part of the specimen. Is referred to as a “second scanning optical system”.

一般的に、共焦点顕微鏡では、標本上の焦点と共役な焦点を検出器前に設けて、そこにピンホールを設置することにより、標本の深さ方向の分解能を1.22λ/NAとして、通常の顕微鏡観察よりも小さくする共焦点効果を利用している。この共焦点効果により、分解能があがるので、標本上を走査している断面の切れの良い画像(すなわち、深さ方向に対して薄いスライス像を得られる画像)が得られる。   In general, in a confocal microscope, a focal point conjugate with the focal point on the specimen is provided in front of the detector, and a pinhole is provided there, so that the resolution in the depth direction of the specimen is 1.22λ / NA. The confocal effect is used to make it smaller than normal microscope observation. Since the resolution increases due to the confocal effect, an image having a good cross section scanned on the specimen (that is, an image capable of obtaining a thin slice image in the depth direction) can be obtained.

なお、高速に画像を取るときや暗い標本では、ピンホールを開いて共焦点効果を弱め、蛍光の分解能を犠牲にして画像を明るくすることが可能である。   When taking images at high speed or in dark specimens, it is possible to open the pinhole to weaken the confocal effect and brighten the image at the expense of fluorescence resolution.

この様に、共焦点顕微鏡は、ピンホールを開いて深さ方向の分解能を落とすことにより、深さ方向の情報を得ることができる。しかし、標本上での焦点深度は対物レンズに入射するコヒーレント光の光束径で決定されるので、ピンホールで、焦点深度を変えることは不可能である。   Thus, the confocal microscope can obtain information in the depth direction by opening the pinhole and reducing the resolution in the depth direction. However, since the depth of focus on the sample is determined by the beam diameter of coherent light incident on the objective lens, it is impossible to change the depth of focus with a pinhole.

一方、顕微鏡による標本への照明としてケーラー照明がよく用いられている。このケーラー照明による標本断面の厚さ方向の照明は、一様に近い励起をしている。   On the other hand, Koehler illumination is often used as illumination of a specimen by a microscope. The illumination in the thickness direction of the specimen cross section by this Koehler illumination is excited almost uniformly.

上記のような従来の共焦点顕微鏡において、2つのレーザ走査光路と一つの対物レンズを用いて装置を実現した場合に、標本に刺激を加えるレーザ光と画像取得のためのレーザ光の標本面上での深さ方向の励起光強度分布が、波長の差しか生じず、ほぼ同一となってしまう。   In the conventional confocal microscope as described above, when the apparatus is realized by using two laser scanning optical paths and one objective lens, the laser beam for stimulating the sample and the laser beam for image acquisition on the sample surface The excitation light intensity distribution in the depth direction at is substantially the same without any difference in wavelength.

本発明は、標本に刺激を加えるレーザ光と画像取得のためのレーザ光(インコヒーレント光でも良い)との標本面上での深さ方向の励起光強度分布を変化させることができる共焦点顕微鏡装置及び共焦点顕微鏡装置を用いた観察方法を提供することを目的とする。   The present invention provides a confocal microscope capable of changing the excitation light intensity distribution in the depth direction on the sample surface between a laser beam for stimulating the sample and a laser beam for image acquisition (or incoherent light). An object is to provide an observation method using the apparatus and the confocal microscope apparatus.

本発明の一局面に係る顕微鏡装置は、コヒーレントではない光源から出力された光によって対物レンズを介して標本を走査し、前記標本からの蛍光を前記対物レンズを介して検出する第1の走査光学系と、レーザ光源から出力されたレーザ光を標本の特定の部位に照射し、特異現象を発現させるための第2の走査光学系とを具備し、前記第1の走査光学系は、共焦点効果を得るための回転ディスクを更に具備し、前記コヒーレントではない光源から出力された光は、前記回転ディスクを介して標本を走査し、前記蛍光は、前記回転ディスクを介して検出され、前記第2の走査光学系は回転ディスクを介さずに前記レーザ光を前記標本の特定部位に集光し、前記第1の光学系及び前記第2の走査光学系の焦点面は同じ深さ位置にあり、前記第2の走査光学系は、前記レーザ光をXY方向に任意に偏向する走査手段によってそのレーザ光を前記特定の部位に照射することを特徴とする。 A microscope apparatus according to one aspect of the present invention scans a sample through an objective lens with light output from a non-coherent light source, and detects fluorescence from the sample through the objective lens. And a second scanning optical system for irradiating a specific part of the specimen with a laser beam output from the laser light source and causing a specific phenomenon to occur. The first scanning optical system includes a confocal lens. A rotating disk for obtaining an effect; light output from the non-coherent light source scans the sample through the rotating disk; the fluorescence is detected through the rotating disk; The second scanning optical system focuses the laser beam on a specific part of the specimen without using a rotating disk, and the focal planes of the first optical system and the second scanning optical system are at the same depth. The second Scanning optical system, and then irradiating the laser beam to the specific site by scanning means for deflecting any said laser beam in the XY direction.

本発明によれば、刺激を与える光学系と画像を取得する励起光の標本面での深さ方向の強度分布を変えることにより、異なる3次元空間の動的解析が可能になる。 According to the present invention, it is possible to dynamically analyze different three-dimensional spaces by changing the intensity distribution in the depth direction on the specimen surface of the excitation light for acquiring images and the excitation light for acquiring images.

本発明の第1の実施形態に係る共焦点顕微鏡装置の概略構成図。 1 is a schematic configuration diagram of a confocal microscope apparatus according to a first embodiment of the present invention. 第1のビーム径可変機構と第2のビーム径可変機構の構成例を示す図。 The figure which shows the structural example of a 1st beam diameter variable mechanism and a 2nd beam diameter variable mechanism. 本発明の第2の実施形態に係る共焦点顕微鏡装置の概略構成図。 The schematic block diagram of the confocal microscope apparatus which concerns on the 2nd Embodiment of this invention. 本発明に適用される回転ディスクの一例を示す図。 The figure which shows an example of the rotating disk applied to this invention. 神経組織の観察を模式的に示した図。 The figure which showed the observation of the nerve tissue typically.

図面を参照して本発明の実施の形態を説明する。 Embodiments of the present invention will be described with reference to the drawings.

(第1の実施形態)
図1は、本発明の第1の実施形態に係る共焦点顕微鏡装置の概略構成図である。

図1において、共焦点顕微鏡装置は、第1のレーザ光源101からのレーザ光で標本134の焦点面上を走査する観察用(又は、画像取得用)の第1の走査光学系100と、第2のレーザ光源201から出力されるレーザ光を標本134の任意の位置に照射してケージド試薬を開裂させるため(すなわち、標本刺激用)の第2の走査光学系200とを備えている。 In FIG. 1, the confocal microscope apparatus includes a first scanning optical system 100 for observation (or image acquisition) that scans the focal plane of the sample 134 with a laser beam from a first laser light source 101, and a first It is provided with a second scanning optical system 200 for cleaving the caged reagent (that is, for stimulating the sample) by irradiating an arbitrary position of the sample 134 with the laser light output from the laser light source 201 of 2. 第1の走査光学系100の光路と第2の走査光学系200の光路とはダイクロイックミラー120で一致している。 The optical path of the first scanning optical system 100 and the optical path of the second scanning optical system 200 coincide with each other by the dichroic mirror 120. これにより、第1の走査光学系100と第2の走査光学系200とが、1つの対物レンズ132を共用している。 As a result, the first scanning optical system 100 and the second scanning optical system 200 share one objective lens 132. (First embodiment) (First embodiment)
FIG. 1 is a schematic configuration diagram of a confocal microscope apparatus according to the first embodiment of the present invention. FIG. 1 is a schematic configuration diagram of a confocal microscope apparatus according to the first embodiment of the present invention.
In FIG. 1, the confocal microscope apparatus includes a first scanning optical system 100 for observation (or image acquisition) that scans the focal plane of a specimen 134 with a laser beam from a first laser light source 101, and a first scanning optical system 100. And a second scanning optical system 200 for cleaving the caged reagent by irradiating laser light output from the second laser light source 201 to an arbitrary position of the specimen 134 (that is, for specimen stimulation). The optical path of the first scanning optical system 100 and the optical path of the second scanning optical system 200 coincide with each other at the dichroic mirror 120. As a result, the first scanning optical system 100 and the second scanning optical system 200 share one objective lens 132. In FIG. 1, the confocal microscope apparatus includes a first scanning optical system 100 for observation (or image acquisition) that scans the focal plane of a specimen 134 with a laser beam from a first laser light source 101, and a first scanning optical system 100. And a second scanning optical system 200 for cleaving the caged reagent by irradiating laser light output from the second laser light source 201 to an arbitrary position of the specimen 134 (that is, for specimen stimulation). The optical path of the first scanning optical system 100 and the optical path of the second scanning optical system 200 coincide with each other at the dichroic mirror 120. As a result, the first scanning optical system 100 and the second scanning optical system 200 share one objective lens 132.

第1の走査光学系100と第2の走査光学系200において、第1のレーザ光源101から出力されたコヒーレント光は第1のビーム径可変機構102及び第1の走査光学ユニット104を介してダイクロイックミラー120に至る。また、第2のレーザ光源201出力されたコヒーレント光は第2のビーム径可変機構202及び第2の走査光学ユニット203を介してダイクロイックミラー120に至る。   In the first scanning optical system 100 and the second scanning optical system 200, the coherent light output from the first laser light source 101 is dichroic via the first beam diameter variable mechanism 102 and the first scanning optical unit 104. The mirror 120 is reached. Further, the coherent light output from the second laser light source 201 reaches the dichroic mirror 120 via the second beam diameter variable mechanism 202 and the second scanning optical unit 203.

また、第1のビーム径可変機構102と第2のビーム径可変機構202は、励起光強度分布算出手段160に電気的又は間接的に接続されている。これにより、励起光強度分布算出手段160が第1のビーム径可変機構102と第2のビーム径可変機構202から出力されるビーム径の情報を得ることができる。   Further, the first beam diameter variable mechanism 102 and the second beam diameter variable mechanism 202 are electrically or indirectly connected to the excitation light intensity distribution calculating means 160. Thereby, the excitation light intensity distribution calculating unit 160 can obtain information on the beam diameters output from the first beam diameter variable mechanism 102 and the second beam diameter variable mechanism 202.

第1のビーム径可変機構102と第2のビーム径可変機構202は、例えば、図2に示すように、ビームエクスパンダーなどのように光束径を変化できるものが、回転するターレツトに複数個(本)装着されているもので良い。又は、第1のビーム径可変機構102と第2のビーム径可変機構202として、複数のレンズ等の光学素子を組み合わせて、レーザのコヒーレントを保ったまま光束径を変化させるような機構(例えば、ズーム機構)を採用しても良い。   For example, as shown in FIG. 2, the first beam diameter variable mechanism 102 and the second beam diameter variable mechanism 202 have a plurality of elements (such as a beam expander) that can change the beam diameter in a rotating turret. Book) What is installed may be used. Alternatively, as the first beam diameter variable mechanism 102 and the second beam diameter variable mechanism 202, a mechanism that changes the beam diameter while maintaining the coherence of the laser by combining optical elements such as a plurality of lenses (for example, A zoom mechanism may be employed.

上記のように構成された第1の実施形態に係る共焦点顕微鏡装置の動作について説明する。 The operation of the confocal microscope apparatus according to the first embodiment configured as described above will be described.

第1の走査光学系100と第2の走査光学系200は、標本134の任意の(所望の)位置にコヒーレント光を照射するために用いる。具体的には、以下の通りである。 The first scanning optical system 100 and the second scanning optical system 200 are used to irradiate an arbitrary (desired) position of the specimen 134 with coherent light. Specifically, it is as follows.

すなわち、第1のレーザ光源101と第2のレーザ光源201から発せられたコヒーレント光は、それぞれ、第1のビーム径可変機構102と第2のビーム径可変機構202で光束径が可変(調整)される。   That is, the coherent light emitted from the first laser light source 101 and the second laser light source 201 is variable (adjusted) by the first beam diameter variable mechanism 102 and the second beam diameter variable mechanism 202, respectively. Is done.

第1のビーム径可変機構102からの出力光は、ダイクロイックミラー150を通過して、第1の走査光学ユニット104の各走査ミラー104a、104bによってXY方向に任意に偏向される。偏向された光は、リレーレンズ105を透過した後に、ミラー106で反射されて、ダイクロイックミラー120に入射する。一方、第2のビーム径可変機構202からの出力光は、第2の走査光学ユニット203の各走査ミラー203a、203bによってXY方向に任意に偏向される。偏向された光は、リレーレンズ204を透過してダイクロイックミラー120に入射し、ダイクロイックミラー120で光路が偏向される。   The output light from the first beam diameter varying mechanism 102 passes through the dichroic mirror 150 and is arbitrarily deflected in the XY directions by the scanning mirrors 104a and 104b of the first scanning optical unit 104. The deflected light passes through the relay lens 105, is reflected by the mirror 106, and enters the dichroic mirror 120. On the other hand, the output light from the second beam diameter variable mechanism 202 is arbitrarily deflected in the XY directions by the scanning mirrors 203 a and 203 b of the second scanning optical unit 203. The deflected light passes through the relay lens 204 and enters the dichroic mirror 120, and the optical path is deflected by the dichroic mirror 120.

そして、ダイクロイックミラー120からのコヒーレント光は、結像レンズ130に入射する。なお、第1のビーム径可変機構102と第2のビーム径可変機構202で第1のレーザ光源からのレーザビームと第2のレーザ光源からのレーザビームの光束径を対物レンズ132の瞳径に対して変化させることにより、各走査光学系に対応する標本134面における深さ方向の励起光分布(そして/又は強度分布)の幅を変化させることができる。   The coherent light from the dichroic mirror 120 enters the imaging lens 130. Note that the beam diameters of the laser beam from the first laser light source and the laser beam from the second laser light source are changed to the pupil diameter of the objective lens 132 by the first beam diameter variable mechanism 102 and the second beam diameter variable mechanism 202. In contrast, the width of the excitation light distribution (and / or intensity distribution) in the depth direction on the surface of the specimen 134 corresponding to each scanning optical system can be changed.

結像レンズ130を透過した光は、対物レンズ132に至り、この対物レンズ132を透過して、ステージ136上に載置された標本134の任意の断面138に集光される。なお、ステージ136は、XY平面方向と高さ方向(Z軸方向:図1における矢印方向)に移動可能である。   The light transmitted through the imaging lens 130 reaches the objective lens 132, passes through the objective lens 132, and is condensed on an arbitrary cross section 138 of the specimen 134 placed on the stage 136. The stage 136 is movable in the XY plane direction and the height direction (Z-axis direction: arrow direction in FIG. 1).

上記のように、標本134を走査する場合において、用途に応じて、各走査ミラー203a、203bによってある範囲を走査してもよく、又静止させてスポット的に照射させてもよい。更に、各走査ミラー203a、203bを瞬間的にスキップ作動させることで瞬時に複数の任意の位置にスポット的に照射させてもよい。一方、第1のレーザ光源101から発せられたコヒーレント光は、上記のように、ダイクロイックミラー150を透過し、第1の走査光学ユニット104の各走査ミラー104a、104bによって偏向される。   As described above, when scanning the specimen 134, a range may be scanned by the scanning mirrors 203a and 203b depending on the application, or the sample 134 may be stationary and irradiated spotwise. Furthermore, a plurality of arbitrary positions may be spot-irradiated instantaneously by instantaneously skipping each scanning mirror 203a, 203b. On the other hand, the coherent light emitted from the first laser light source 101 passes through the dichroic mirror 150 and is deflected by the scanning mirrors 104 a and 104 b of the first scanning optical unit 104 as described above.

第1の走査光学系100によって標本134に光が照射されると、この光により蛍光指示薬が励起され、蛍光が発せられる。   When the specimen 134 is irradiated with light by the first scanning optical system 100, the fluorescent indicator is excited by this light and emits fluorescence.

この標本134からの蛍光は、標本134への入射光路とは逆方向に、対物レンズ132から結像レンズ130、ダイクロイックミラー120、第1の走査光学ユニット104、リレーレンズ103、各走査ミラー104b、104aを通ってダイクロイックミラー150に到達し、このダイクロイックミラー150で反射して測光フィルタ140に入射する。   The fluorescence from the specimen 134 is transmitted from the objective lens 132 to the imaging lens 130, the dichroic mirror 120, the first scanning optical unit 104, the relay lens 103, each scanning mirror 104b, in the direction opposite to the incident optical path to the specimen 134. The light reaches the dichroic mirror 150 through 104 a, is reflected by the dichroic mirror 150, and enters the photometric filter 140.

この測光フィルタ140に入射した光は、標本134からの蛍光波長のみが選択され、標本134からの蛍光波長のみを有する光がレンズ142によってピンホール144面に結像される。このピンホール144を通過した蛍光は、光電変換素子146によって計測される。   For the light incident on the photometric filter 140, only the fluorescence wavelength from the specimen 134 is selected, and light having only the fluorescence wavelength from the specimen 134 is imaged on the surface of the pinhole 144 by the lens 142. The fluorescence that has passed through the pinhole 144 is measured by the photoelectric conversion element 146.

励起光強度分布算出手段160は、第1のビーム径可変機構102と第2のビーム径可変機構202より出力されるビーム径及び現在使用されている対物レンズの性能(仕様)の情報を入力して標本面上での励起光強度分布を算出したり、既に記憶されている値を図では示されていないコンピュータ又はディスプレイなどのインターフェースに出力したりするなどの機能を有する。   The excitation light intensity distribution calculating means 160 inputs information on the beam diameters output from the first beam diameter variable mechanism 102 and the second beam diameter variable mechanism 202 and the performance (specifications) of the objective lens currently used. Thus, it has functions such as calculating the excitation light intensity distribution on the sample surface and outputting the already stored values to an interface such as a computer or a display not shown in the figure.

上記のような、本発明の第1の実施形態に係る共焦点顕微鏡装置によれば、第1の走査光学系100により標本画像を観察・記録している最中に、第2の走査光学系200によりコヒーレント光を標本134に照射することによって、第2の走査光学系200によるコヒーレント光照射によって引き起こされる標本134の動的特性(化学反応)などを第1の走査光学系100で調査できる。   According to the confocal microscope apparatus according to the first embodiment of the present invention as described above, the second scanning optical system is in the process of observing and recording the sample image by the first scanning optical system 100. By irradiating the specimen 134 with coherent light by 200, the dynamic characteristics (chemical reaction) of the specimen 134 caused by the coherent light irradiation by the second scanning optical system 200 can be investigated by the first scanning optical system 100.

この場合において、第1の実施形態では、第1の走査光学系100と第2の走査光学系200による標本面上での深さ方向の励起光分布を、第1のビーム径可変機構102と第2のビーム径可変機構202によって独立に設定できる。従って、例えば、第2の走査光学系によって標本を刺激している範囲の励起光分布の幅が狭い場合であって、その刺激によって標本の厚み方向に対して広い領域に影響があった場合でも、第1の走査光学系の励起光分布の幅を広くすることによって、その様子を観察することが可能である。   In this case, in the first embodiment, the excitation light distribution in the depth direction on the specimen surface by the first scanning optical system 100 and the second scanning optical system 200 is expressed as the first beam diameter variable mechanism 102. It can be set independently by the second beam diameter variable mechanism 202. Therefore, for example, even when the width of the excitation light distribution in the range in which the specimen is stimulated by the second scanning optical system is narrow and the stimulation affects a wide area in the thickness direction of the specimen. It is possible to observe the situation by increasing the width of the excitation light distribution of the first scanning optical system.

また、上記とは逆に、第2の走査光学系で標本の厚み方向に対して広範囲な部分を刺激して、第1の走査光学系では深さ方向の励起光分布の幅を狭くすることにより、標本の断面138を高分解能で観察することもできる。   In contrast to the above, the second scanning optical system stimulates a wide range with respect to the specimen thickness direction, and the first scanning optical system narrows the width of the excitation light distribution in the depth direction. Thus, the cross section 138 of the specimen can be observed with high resolution.

第1の実施形態において、第1のレーザ光源101として、IRパルスレーザを用いて、2光子吸収により蛍光画像を取得する構成としても良い。この場合において、2光子吸収現象は結像位置でのみ発生するので、ピンホール144は理論的には不要になる。また、ダイクロイックミラー150は、IRレーザを透過し、可視蛍光を反射して光電変換素子146に導くために、短波長反射の特性を持つものとなる。また、第1のビーム径可変機構102は使用しない構成とする。   In the first embodiment, the first laser light source 101 may be configured to acquire a fluorescent image by two-photon absorption using an IR pulse laser. In this case, the two-photon absorption phenomenon occurs only at the imaging position, so the pinhole 144 is theoretically unnecessary. In addition, the dichroic mirror 150 transmits the IR laser, reflects visible fluorescence, and guides it to the photoelectric conversion element 146. Therefore, the dichroic mirror 150 has a short wavelength reflection characteristic. The first beam diameter variable mechanism 102 is not used.

上記のように、第1のレーザ光源101として、IRパルスレーザを用いることにより、第1の走査光学系の構成を簡略化することができる。加えて、第1のビーム径可変機構102を使用しない場合であっても、走査光学系100の標本面上での深さ方向の励起光分布の幅は、2光子吸収現象により第2の走査光学系200の深さ方向の励起光分布の幅よりも狭くなる。また、標本を刺激する標本の厚みを変化させたい場合は、第2のビーム径可変機構202によって第2の走査光学系200の励起光分布の幅を狭くすることができる。   As described above, by using an IR pulse laser as the first laser light source 101, the configuration of the first scanning optical system can be simplified. In addition, even when the first beam diameter varying mechanism 102 is not used, the width of the excitation light distribution in the depth direction on the sample surface of the scanning optical system 100 is the second scanning due to the two-photon absorption phenomenon. It becomes narrower than the width of the excitation light distribution in the depth direction of the optical system 200. Further, when it is desired to change the thickness of the specimen that stimulates the specimen, the width of the excitation light distribution of the second scanning optical system 200 can be narrowed by the second beam diameter variable mechanism 202.

(第2の実施形態)
図3を参照して、本発明の第2の実施形態に係る共焦点顕微鏡装置を説明する。図3は、本発明の第2の実施形態に係る共焦点顕微鏡装置の概略構成図である。図3において、第2の走査光学系200は、第1の実施形態と同じであるので、同じ符号を付し、詳細な説明は省略する。
(Second Embodiment)
A confocal microscope apparatus according to a second embodiment of the present invention will be described with reference to FIG. FIG. 3 is a schematic configuration diagram of a confocal microscope apparatus according to the second embodiment of the present invention. In FIG. 3, the second scanning optical system 200 is the same as that of the first embodiment, so the same reference numerals are given and detailed description is omitted.

第2の実施形態において、第1の走査光学系100′は、光源301として水銀光源やハロゲン光源(これらの光源を「ランプ」とも称する)やLED光源等のコヒーレントではない光源を有している。光源301から出射される光の光路上には、光学レンズ302や、偏光板303や、偏光ビームスプリッター(PBS)304が配置されている。   In the second embodiment, the first scanning optical system 100 ′ has a non-coherent light source such as a mercury light source, a halogen light source (these light sources are also referred to as “lamps”), and an LED light source as the light source 301. . On the optical path of light emitted from the light source 301, an optical lens 302, a polarizing plate 303, and a polarizing beam splitter (PBS) 304 are arranged.

PBS304の反射光路上には、回転ディスク305と、第1結像レンズ307と、1/4波長板308と、対物レンズ309とが配置されており、これらを介して光源からの光が標本310に入射する。   A rotating disk 305, a first imaging lens 307, a quarter-wave plate 308, and an objective lens 309 are disposed on the reflected light path of the PBS 304, and light from the light source passes through the sample 310 via these. Is incident on.

回転ディスク305は、回転軸306を介して図示しないモータの軸に連結されており、一定の回転速度で回転するようになっている。なお、回転ディスク305は、例えば、直線状の光が通過する通過部分と光を遮蔽する遮蔽部分が交互に並んで配置されている。そして、遮蔽部分のラインの幅は、通過部分より広く、例えば、遮蔽部分と通過部分のラインの幅の比は、1:9になっている(図4参照)。   The rotating disk 305 is connected to a motor shaft (not shown) via a rotating shaft 306 and rotates at a constant rotational speed. Note that the rotating disk 305 has, for example, a passing portion through which linear light passes and a shielding portion that blocks light alternately arranged. The line width of the shielding part is wider than that of the passage part. For example, the ratio of the line width of the shielding part and the passage part is 1: 9 (see FIG. 4).

また、光が透過する部分の幅をWとすれば、ピンホールの場合と同じく、試料像がディスクに投影される倍率をM、光の波長をλ、対物レンズの開口率をNAとして、
W=kλM/NA
となる。ここで、kは係数であり、k=0.5〜1程度の値がよく使われる。
If the width of the light transmitting portion is W, as in the case of the pinhole, the magnification at which the sample image is projected onto the disk is M, the wavelength of the light is λ, and the aperture ratio of the objective lens is NA.
W = kλM / NA
It becomes. Here, k is a coefficient, and a value of about k = 0.5 to 1 is often used.

また、PBS304の透過光路上には、第2の結像レンズ311を介してCCDカメラ312が配置されている。CCDカメラ312には、CCDカメラ312で撮像した画像を観察するモニタ313が接続されている。 In addition, a CCD camera 312 is disposed on the transmission optical path of the PBS 304 via a second imaging lens 311. Connected to the CCD camera 312 is a monitor 313 for observing an image captured by the CCD camera 312.

上記のように構成された第2の実施形態に係る共焦点顕微鏡装置の動作を説明する。 The operation of the confocal microscope apparatus according to the second embodiment configured as described above will be described.

光源301から出射された光は、光学レンズ302を通って、偏光板303で、所定の偏光のみの直線偏光となって、PBS304に入射する。PBS304は、偏光板を透過してきた方向の偏光は反射し、それに垂直な方向の偏光は透過する。   The light emitted from the light source 301 passes through the optical lens 302, becomes linearly polarized light of only predetermined polarization at the polarizing plate 303, and enters the PBS 304. The PBS 304 reflects polarized light in the direction transmitted through the polarizing plate and transmits polarized light in a direction perpendicular thereto.

PBS304で反射された光は、一定の速度で回転する回転ディスク305に入射する。回転ディスク305の透過部分を透過した光は第1の結像レンズ307を通り、1/4波長板308で円偏光となって、対物レンズ309によって結像され、標本310に入射される。   The light reflected by the PBS 304 is incident on a rotating disk 305 that rotates at a constant speed. The light transmitted through the transmission part of the rotating disk 305 passes through the first imaging lens 307, becomes circularly polarized light by the quarter wavelength plate 308, forms an image by the objective lens 309, and enters the sample 310.

標本310で反射された光は、対物レンズ309を通り、1/4波長板308で、入射時と直交した直線偏光となり、第2の結像レンズ311を介して回転ディスク305上に標本310の像を結像する。   The light reflected by the sample 310 passes through the objective lens 309, becomes a linearly polarized light orthogonal to the incident time by the quarter-wave plate 308, and passes through the second imaging lens 311 on the rotating disk 305. Form an image.

回転ディスク305上に結像された像のうち、焦点の合っている成分は、回転ディスク305上の透過部分を通過する。回転ディスク305を通過した成分は、PBS304を透過して、第2結像レンズ311を介してCCDカメラ312に到達し、その結像面(撮像面)に試料像が結像される。   Of the image formed on the rotating disk 305, the focused component passes through the transmission part on the rotating disk 305. The component that has passed through the rotating disk 305 passes through the PBS 304 and reaches the CCD camera 312 via the second imaging lens 311, and a sample image is formed on its imaging surface (imaging surface).

標本310を観察しているときのある瞬間を考えれば、標本310上には図4のように、ある方向にラインが投影されている。   Considering a certain moment when the sample 310 is observed, a line is projected on the sample 310 in a certain direction as shown in FIG.

このような状態で、標本310から反射した光が回転ディスク305上に結像された場合には、標本310について、その焦点が合っている部分は回転ディスク上にラインが投影される。しかし、非合焦部分は回転ディスク305に投影された像がボケているので、非合焦像の大部分はディスクを透過できないことになる。従って、合焦した像のみが回転ディスク305を透過することになる。   In such a state, when the light reflected from the specimen 310 is imaged on the rotating disk 305, a line is projected onto the rotating disk for the portion of the specimen 310 that is in focus. However, since the image projected on the rotating disk 305 is blurred in the out-of-focus portion, most of the out-of-focus image cannot pass through the disc. Therefore, only the focused image passes through the rotating disk 305.

なお、回転ディスク305が回転しない場合には、このままの状態であって、単にサンプルとラインが重なった像である。しかし、回転ディスク305を回転させることによって、透過部分と遮蔽部分からなるラインが標本310上を方向を変えながら移動して行くことになるので、平均化されてライン像は消えて、焦点のあった画像が観察される。従って、CCDカメラ312の露出時間に対して、回転ディスク305の回転が十分速ければ、合焦画像をCCDカメラ312で撮像しモニタ313で観察できることになる。例えば、CCDカメラ312が通常でTVレートならば、露出時間は1/60秒か1/30秒であるから露出時間中に回転ディスク305の回転数を、半回転する程度の1800rpmくらいにすれば良い。   When the rotating disk 305 does not rotate, it remains as it is, and is simply an image in which the sample and the line overlap. However, when the rotating disk 305 is rotated, the line composed of the transmission part and the shielding part moves on the sample 310 while changing the direction, so that the line image is averaged out and the focus is adjusted. The observed image is observed. Therefore, if the rotation of the rotary disk 305 is sufficiently fast with respect to the exposure time of the CCD camera 312, a focused image can be picked up by the CCD camera 312 and observed on the monitor 313. For example, if the CCD camera 312 is normal and the TV rate, the exposure time is 1/60 second or 1/30 second, so if the rotation speed of the rotating disk 305 is set to about 1800 rpm, which is a half rotation during the exposure time. good.

この時における第1の走査光学系100′の標本310面上での深さ方向の励起光分布は、スリットの長手方向では、顕微鏡のケーラー照明の照射分布と同じである。スリットの幅方向では、第2の走査光学系と同じ分布である。   The excitation light distribution in the depth direction on the sample 310 surface of the first scanning optical system 100 ′ at this time is the same as the irradiation distribution of the Koehler illumination of the microscope in the longitudinal direction of the slit. In the slit width direction, the distribution is the same as that of the second scanning optical system.

従って、第1の走査光学系の標本面上での探さ方向の励起光分布は、長手方向と幅方向のものとが合成されたものになる。なお、励起光の深さ方向の強度分布を変更させるには、回転ディスク305のスリット幅及びスリット間隔を変えることで可変可能である。   Accordingly, the excitation light distribution in the search direction on the sample surface of the first scanning optical system is a combination of the longitudinal direction and the width direction. Note that the intensity distribution in the depth direction of the excitation light can be changed by changing the slit width and slit interval of the rotating disk 305.

第2の実施形態では、第1の実施形態で示した第2の走査光学系により照射された光の反応を受けた動的変化を第1の走査光学系100′で検出することで、第1と第2の標本面上での深さ方向の励起光分布が違って出来る。従って、第2の走査光学系200が励起している範囲よりもより広範囲な測定が第1の走査光学系100′で可能である。   In the second embodiment, the first scanning optical system 100 ′ detects a dynamic change in response to the light irradiated by the second scanning optical system shown in the first embodiment. The excitation light distribution in the depth direction on the first and second specimen surfaces can be different. Accordingly, the first scanning optical system 100 ′ can perform measurement over a wider range than the range in which the second scanning optical system 200 is excited.

特に神経系の測定では、標本の厚み方向に伸びている神経の動きを捉えるには、高速に画像を取得する必要がある。通常、共焦点顕微鏡装置では、標本面上での深さ方向の励起光分布の幅が狭いため、標本の厚み方向に伸びていると一度の測定では、画像を捉えることが出来ない。よって、第2の実施の形態のように、標本面上での探さ方向の励起光分布の幅を広くして測定することにより広範囲の画像測定が出来る効果がある。このため、第2の実施形態において、回転ディスク305を省略した構成としても良い。また、回転ディスクは、図4に示したものに限られず、共焦点効果が得られるようなものであれば、どのような形状或いは、構成のものを用いてもよい。例えば、回転ディスクにピンホールが形成されているものであっても良いし、上記の実施の形態のような透過型ではなく、反射型のものとしても良い。   Particularly in the measurement of the nervous system, it is necessary to acquire an image at high speed in order to capture the movement of the nerve extending in the thickness direction of the specimen. Usually, in the confocal microscope apparatus, since the width of the excitation light distribution in the depth direction on the sample surface is narrow, if the sample extends in the thickness direction of the sample, an image cannot be captured by one measurement. Therefore, as in the second embodiment, there is an effect that a wide range of image measurement can be performed by increasing the width of the excitation light distribution in the probe direction on the specimen surface. For this reason, in the second embodiment, the rotating disk 305 may be omitted. Further, the rotating disk is not limited to that shown in FIG. 4 and may be of any shape or configuration as long as a confocal effect can be obtained. For example, a pinhole may be formed in the rotating disk, or a reflective type may be used instead of the transmissive type as in the above embodiment.

また、第2の実施形態では、第2のビーム径可変機構202は必ずしも必要ではないが、第2のビーム径可変機構202があれば、第1の断面と第2の断面の比を変えることが可能であり、画像を取得する範囲と刺激を与える部分の微調整により、実験(そして/又は観察)の自由度が広がる。また、第2のビーム径可変機構202を設ける場合には、第1の実施形態と同様に励起光強度分布算出手段160を設けることが好ましい。   In the second embodiment, the second beam diameter varying mechanism 202 is not necessarily required. However, if the second beam diameter varying mechanism 202 is provided, the ratio between the first cross section and the second cross section can be changed. The degree of freedom of experiment (and / or observation) is expanded by fine-tuning the range for acquiring an image and the portion for giving a stimulus. Further, when the second beam diameter variable mechanism 202 is provided, it is preferable to provide the excitation light intensity distribution calculating means 160 as in the first embodiment.

また、上記の構成において、第1の走査光学系100′をケーラー照明による顕微鏡光学系で構成することにより、より広い励起範囲での画像取得が可能である。なお、この場合には、回転ディスク305は不要になる。   Further, in the above configuration, the first scanning optical system 100 ′ is configured by a microscope optical system using Kohler illumination, so that an image can be acquired in a wider excitation range. In this case, the rotating disk 305 is not necessary.

上記の第2の実施形態において、PBS304をダイクロイックミラーに変更しても良い。このようにした場合には、ダイクロイックミラーで光源からの光を反射させ、標本からの蛍光を通過させることによって、励起光学系と測定光学系の光路を分離できるので、偏光板303は不要になる。   In the second embodiment, the PBS 304 may be changed to a dichroic mirror. In this case, the optical path of the excitation optical system and the measurement optical system can be separated by reflecting the light from the light source by the dichroic mirror and passing the fluorescence from the specimen, so that the polarizing plate 303 is not necessary. .

上記の各実施形態における共焦点顕微鏡装置の用途としては、例えば、細胞における研究分野では、局所的に励起し、励起した部位からの反応を観察する用途がある。   As an application of the confocal microscope apparatus in each of the above-described embodiments, for example, in the field of research in cells, there is an application of locally exciting and observing a reaction from the excited site.

アンケージド(Uncaged)という手法では、局所的に励起することで活性物質の濃度が変化する。その濃度変化を計測する場合に、局所的に励起した部位以外の周辺部分を同時に計測することで、細胞内の機能解析が行なうことが可能である。   In the method called uncaged, the concentration of the active substance is changed by local excitation. When measuring the concentration change, it is possible to analyze the function in the cell by simultaneously measuring the peripheral part other than the locally excited part.

フォトブリーチという手法では、細胞を局所的に励起することによって、当該部位の退色を施す。その部位は、周辺のタンパク質の移動により時間とともに退色した部位が復帰する現象が見られる。従って、局所的に励起した部位と周辺部分との両者での計測が必要になる。   In a technique called photo bleaching, the cell is fading by locally exciting the cells. The site shows a phenomenon in which the site that has faded over time due to the movement of surrounding proteins is restored. Therefore, it is necessary to measure both the locally excited part and the peripheral part.

図5を参照してその一例を示す。図5は神経組織の観察を模式的に示した図である。   An example is shown with reference to FIG. FIG. 5 is a diagram schematically showing the observation of nerve tissue.

例えば細胞体1から細胞体2に軸索3を伝わるイオンを細胞体1に注入されたケージド蛍光色素をプローブとして観察しようとする場合には、まず、細胞体1上の焦点面4に標本を刺激するためのレーザ光を照射する。そして、その後の変化を標本観察用のレーザ光で観察する。しかし、観察用のレーザ光の深さ方向の励起光強度分布は、通常は刺激用のレーザ光と同じ深さの励起光強度分布5となるので、従来では、その分布の範囲内に入っていない軸索3を伝わる蛍光色素は励起光が当たらないため観察できないことがある。これに対して、本発明の各実施形態においては、標本に刺激を加えるレーザ光と画像取得のためのレーザ光との標本面上での深さ方向の励起光強度分布をそれぞれ独立に変化させることができるので、従来の問題を解決できる。   For example, when observing ions transmitted from the cell body 1 to the cell body 2 through the axon 3 using a caged fluorescent dye injected into the cell body 1 as a probe, first, a specimen is placed on the focal plane 4 on the cell body 1. Irradiate laser light for stimulation. Then, the subsequent change is observed with a laser beam for sample observation. However, since the excitation light intensity distribution in the depth direction of the observation laser light is normally the excitation light intensity distribution 5 having the same depth as that of the stimulation laser light, conventionally, it is within the range of the distribution. The fluorescent dye that travels through the axon 3 may not be observed because the excitation light does not strike. On the other hand, in each embodiment of the present invention, the excitation light intensity distribution in the depth direction on the sample surface of the laser beam for stimulating the sample and the laser beam for image acquisition is changed independently. Can solve the conventional problems.

上記の各実施形態から下記の発明が抽出される。なお、本発明は、上記の発明の実施の形態に限定されるものではない。本発明の要旨を変更しない範囲で種々変形して実施できるのは勿論である。   The following invention is extracted from each of the above embodiments. The present invention is not limited to the above-described embodiments. Of course, various modifications can be made without departing from the scope of the present invention.

本発明の第1局面に係る共焦点顕微鏡装置は、第1のレーザ光源からのレーザ光で標本の走査画像を得るための第1の走査光学系と、前記第1のレーザ光源とは異なる第2のレーザ光源からのレーザ光で前記標本の特定部位を走査して、特異現象を発現させるための第2の走査光学系と、前記第1の走査光学系と前記第2の走査光学系の少なくとも一方のレーザ光のビーム径を変化させることができるビーム径可変機構とを具備することを特徴とする。レーザ光学系とレーザ走査型顕微鏡の組合せによる、標本面上での深さ方向の励起強度分布の違いによる測定の幅を変えることが可能である。具体的には、以下の通りである。   A confocal microscope apparatus according to a first aspect of the present invention is different from a first scanning optical system for obtaining a scanned image of a specimen with laser light from a first laser light source and the first laser light source. A second scanning optical system for scanning a specific part of the specimen with laser light from the two laser light sources to develop a specific phenomenon, and the first scanning optical system and the second scanning optical system. And a beam diameter variable mechanism capable of changing the beam diameter of at least one of the laser beams. By combining a laser optical system and a laser scanning microscope, it is possible to change the width of measurement due to the difference in the excitation intensity distribution in the depth direction on the specimen surface. Specifically, it is as follows.

従来においては、標本の動的解析をする場合は、刺激を加える範囲と画像を取得する範囲が異なることはもとより、刺激を加えるレーザ光の標本面上での深さ方向の励起光強度分布と画像を取得するレーザ光の深さ方向の励起光強度分布を互いに異なるようにしたいことがある。その上で、深さ方向の励起光強度分布の幅を意図的に狭くしたいこともある。   Conventionally, when performing dynamic analysis of a specimen, the excitation light intensity distribution in the depth direction on the specimen surface of the laser light to which the stimulus is applied is different from the area where the stimulus is applied and the area where the image is acquired. In some cases, it is desired to make the excitation light intensity distributions in the depth direction of the laser light for acquiring images different from each other. In addition, the width of the excitation light intensity distribution in the depth direction may be intentionally narrowed.

本発明は、上記各実施の形態に限ることなく、その他、実施段階ではその要旨を逸脱しない範囲で種々の変形を実施し得ることが可能である。さらに、上記各実施形態には、種々の段階の発明が含まれており、開示される複数の構成要件における適宜な組合せにより種々の発明が抽出され得る。   The present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the scope of the invention at the stage of implementation. Further, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements.

すなわち、例えば、上記の各実施形態から下記の発明が抽出できる。なお、下記の各発明は単独で適用しても良いし、適宜組み合わせて適用しても良い。   That is, for example, the following invention can be extracted from each of the above embodiments. Each of the following inventions may be applied alone or in appropriate combination.

本発明の第1局面に係る共焦点顕微鏡装置は、各走査光学系のレーザ光の射出口に、レーザ光の光束径を変化させるビーム径可変機構を備えている。このビーム径可変機構によってレーザ光の光束径を小さくした場合、対物レンズの開口数は光束径が大きい場合に比べて小さくなる。その結果、対物レンズを交換しなくても標本面上で探さ方向の励起光強度分布の幅が狭くなる。更に、ビーム径可変機構を各光学系に備えることで、各光学系の標本面上での探さ方向の励起光強度分布を独立に変化させることができる。更にその標本面上での深さ方向の励起光分布の幅を意図的に変えることが可能である。   The confocal microscope apparatus according to the first aspect of the present invention includes a beam diameter variable mechanism that changes the beam diameter of the laser light at the laser light exit of each scanning optical system. When the beam diameter of the laser beam is reduced by this beam diameter variable mechanism, the numerical aperture of the objective lens is smaller than that when the beam diameter is large. As a result, the width of the excitation light intensity distribution in the searching direction on the sample surface is narrowed without exchanging the objective lens. Furthermore, by providing each optical system with a beam diameter variable mechanism, the excitation light intensity distribution in the probe direction on the sample surface of each optical system can be changed independently. Further, it is possible to intentionally change the width of the excitation light distribution in the depth direction on the specimen surface.

本発明の第2局面に係る共焦点顕微鏡装置は、コヒーレントではない光源から出力された光によって対物レンズを介して標本を走査し、前記標本からの蛍光を前記対物レンズを介して検出する第1の走査光学系と、レーザ光源から出力されたレーザ光を標本の特定の部位に照射し、特異現象を発現させるための第2の走査光学系とを具備し、前記第1の走査光学系は、共焦点効果を得るための回転ディスクを更に具備し、前記インコヒーレントな光は、前記回転ディスクを介して標本を走査し、前記蛍光は、前記回転ディスクを介して検出されることを特徴とする。レーザ光学系とディスクタイプの共焦点顕微鏡装置の組合せによる標本面上での探さ方向の励起強度分布の違いによる測定の幅を変えることが可能である。   A confocal microscope apparatus according to a second aspect of the present invention scans a sample through an objective lens with light output from a light source that is not coherent, and detects fluorescence from the sample through the objective lens. And a second scanning optical system for irradiating a specific portion of the specimen with a laser beam output from a laser light source and causing a specific phenomenon to occur, the first scanning optical system comprising: A rotating disk for obtaining a confocal effect, wherein the incoherent light scans a specimen through the rotating disk, and the fluorescence is detected through the rotating disk. To do. By combining a laser optical system and a disk-type confocal microscope apparatus, it is possible to change the measurement width due to the difference in the excitation intensity distribution in the probe direction on the specimen surface.

本発明の第3局面に係る共焦点顕微鏡装置は、コヒーレントではない光源から出力された光によって対物レンズを介して標本を照明し、前記標本からの蛍光を前記対物レンズを介して検出する第1の光学系と、レーザ光源から出力されたレーザ光を標本の特定の部位に照射し、特異現象を発現させるための第2の走査光学系とを具備することを特徴とする。レーザ光学系とケーラー照明における顕微鏡の組合せによる標本面上での深さ方向の励起強度分布の違いによる測定の幅を変えることが可能である。   A confocal microscope apparatus according to a third aspect of the present invention illuminates a specimen through an objective lens with light output from a light source that is not coherent, and detects fluorescence from the specimen through the objective lens. And a second scanning optical system for irradiating a specific part of the specimen with a laser beam output from a laser light source and causing a specific phenomenon to occur. It is possible to change the width of the measurement due to the difference in the excitation intensity distribution in the depth direction on the specimen surface by the combination of the laser optical system and the microscope in Koehler illumination.

上記の共焦点顕微鏡装置の好ましい実施態様は以下のとおりである。なお、以下の各実施態様は、単独で適用しても良いし、適宜組み合わせて適用しても良い。
(1) 前記第2の走査光学系が前記レーザ光源のレーザ光のビーム径を変化させるビーム径可変機構を更に備えること。
(2) 前記ビーム径可変機構より出力されるレーザ光のビーム径から標本面上での深さ方向の励起光強度分布を算出し、又は記憶する励起光強度分布算出手段を更に具備すること。
(3) 前記第1のレーザ光源はIRパルスレーザであり、前記ビーム可変機構は、前記第2の走査光学系に設けられていること。
(4) 前記ビーム径可変機構から出力されたレーザ光の標本面上での深さ方向の強度分布を算出する深さ方向強度分布算出手段を更に備えること。 (4) Further provided with a depth direction intensity distribution calculation means for calculating the intensity distribution in the depth direction on the sample surface of the laser beam output from the beam diameter variable mechanism.
(5) 前記コヒーレントではない光源は、ランプもしくはLED光源であること。 (5) The non-coherent light source shall be a lamp or LED light source. A preferred embodiment of the confocal microscope apparatus is as follows. In addition, each following embodiment may be applied independently and may be applied in combination as appropriate. A preferred embodiment of the confocal microscope apparatus is as follows. In addition, each following embodiment may be applied independently and may be applied in combination as appropriate.
(1) The second scanning optical system further includes a beam diameter variable mechanism that changes a beam diameter of the laser light of the laser light source. (1) The second scanning optical system further includes a beam diameter variable mechanism that changes a beam diameter of the laser light of the laser light source.
(2) It further comprises excitation light intensity distribution calculating means for calculating or storing the excitation light intensity distribution in the depth direction on the specimen surface from the beam diameter of the laser light output from the beam diameter variable mechanism. (2) It further excitation light intensity distribution calculating means for calculating or storing the excitation light intensity distribution in the depth direction on the specimen surface from the beam diameter of the laser light output from the beam diameter variable mechanism.
(3) The first laser light source is an IR pulse laser, and the beam variable mechanism is provided in the second scanning optical system. (3) The first laser light source is an IR pulse laser, and the beam variable mechanism is provided in the second scanning optical system.
(4) It further includes depth direction intensity distribution calculating means for calculating the intensity distribution in the depth direction on the sample surface of the laser beam output from the beam diameter varying mechanism. (4) It further includes depth direction intensity distribution calculating means for calculating the intensity distribution in the depth direction on the sample surface of the laser beam output from the beam diameter varying mechanism.
(5) The non-coherent light source is a lamp or an LED light source. (5) The non-coherent light source is a lamp or an LED light source.

なお、本発明は、上記の共焦点顕微鏡装置を用いた観察方法としても成立する。
また、例えば各実施形態に示される全構成要件から幾つかの構成要件が削除されても、発明が解決しようとする課題の欄で述べた課題が解決でき、発明の効果で述べられている効果が得られる場合には、この構成要件が削除された構成が発明として抽出され得る。 Further, for example, even if some constituent requirements are deleted from all the constituent requirements shown in each embodiment, the problems described in the column of the problems to be solved by the invention can be solved, and the effects described in the effect of the invention can be solved. If is obtained, a configuration in which this configuration requirement is deleted can be extracted as an invention. In addition, this invention is materialized also as an observation method using said confocal microscope apparatus. In addition, this invention is materialized also as an observation method using said confocal microscope apparatus.
In addition, for example, even if some structural requirements are deleted from all the structural requirements shown in each embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and the effect described in the effect of the invention Can be obtained as an invention. In addition, for example, even if some structural requirements are deleted from all the structural requirements shown in each embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and the effect described in the effect of the invention Can be obtained as an invention.

100、100′…第1の走査光学系、101…第1のレーザ光源、102…第1のビーム径可変機構、103…リレーレンズ、104…第1の走査光学ユニット、104a、104b…走査ミラー、105…リレーレンズ、106…ミラー、120…ダイクロイックミラー、130…結像レンズ、132…対物レンズ、134…標本、136…ステージ、140…測光フィルタ、142…レンズ、144…ピンホール、146…光電変換素子、150…ダイクロイックミラー、160…励起光強度分布算出手段、200…第2の走査光学系、201…第2のレーザ光源、202…第2のビーム径可変機構、203…第2の走査光学ユニット、203a、203b…走査ミラー、204…リレーレンズ、301…光源、302…光学レンズ、303…偏光板、304…偏光ビームスプリッター(PBS)、305…回転ディスク、306…回転軸、307…第1の結像レンズ、308…波長板、309…対物レンズ、310…標本、311…第2の結像レンズ、312…CCDカメラ、313…モニタ。   DESCRIPTION OF SYMBOLS 100, 100 '... 1st scanning optical system, 101 ... 1st laser light source, 102 ... 1st beam diameter variable mechanism, 103 ... Relay lens, 104 ... 1st scanning optical unit, 104a, 104b ... Scanning mirror , 105 ... relay lens, 106 ... mirror, 120 ... dichroic mirror, 130 ... imaging lens, 132 ... objective lens, 134 ... sample, 136 ... stage, 140 ... photometric filter, 142 ... lens, 144 ... pinhole, 146 ... Photoelectric conversion element 150 ... Dichroic mirror 160 ... Excitation light intensity distribution calculating means 200 ... Second scanning optical system 201 ... Second laser light source 202 ... Second beam diameter variable mechanism 203 ... Second Scanning optical unit, 203a, 203b ... scanning mirror, 204 ... relay lens, 301 ... light source, 302 ... optical lens, DESCRIPTION OF SYMBOLS 03 ... Polarizing plate 304 ... Polarizing beam splitter (PBS), 305 ... Rotating disk, 306 ... Rotating shaft, 307 ... First imaging lens, 308 ... Wave plate, 309 ... Objective lens, 310 ... Sample, 311 ... First 2 imaging lenses, 312... CCD camera, 313.

Claims (7)

  1. コヒーレントではない光源から出力された光によって対物レンズを介して標本を走査し、前記標本からの蛍光を前記対物レンズを介して検出する第1の走査光学系と、
    レーザ光源から出力されたレーザ光を標本の特定の部位に照射し、特異現象を発現させるための第2の走査光学系とを具備し、
    前記第1の走査光学系は、共焦点効果を得るための回転ディスクを更に具備し、
    前記コヒーレントではない光源から出力された光は、前記回転ディスクを介して標本を走査し、
    前記蛍光は、前記回転ディスクを介して検出され、
    前記第2の走査光学系は回転ディスクを介さずに前記レーザ光を前記標本の特定部位に集光し、
    前記第1の光学系及び前記第2の走査光学系の焦点面は同じ深さ位置にあり、 The focal planes of the first optical system and the second scanning optical system are at the same depth position.
    前記第2の走査光学系は、前記レーザ光をXY方向に任意に偏向する走査手段によってそのレーザ光を前記特定の部位に照射することを特徴とする顕微鏡装置。 The second scanning optical system is a microscope apparatus characterized in that the laser beam is irradiated to the specific portion by a scanning means that arbitrarily deflects the laser beam in the XY directions. A first scanning optical system that scans a sample through an objective lens with light output from a non-coherent light source, and detects fluorescence from the sample through the objective lens; A first scanning optical system that scans a sample through an objective lens with light output from a non-coherent light source, and detects fluorescence from the sample through the objective lens;
    A second scanning optical system for irradiating a specific portion of the specimen with a laser beam output from a laser light source and causing a specific phenomenon to occur, A second scanning optical system for irradiating a specific portion of the specimen with a laser beam output from a laser light source and causing a specific phenomenon to occur,
    The first scanning optical system further comprises a rotating disk for obtaining a confocal effect, The first scanning optical system further a rotating disk for obtaining a confocal effect,
    The light output from the non-coherent light source scans the specimen through the rotating disk, The light output from the non-coherent light source scans the specimen through the rotating disk,
    The fluorescence is detected via the rotating disk; The fluorescence is detected via the rotating disk;
    The second scanning optical system condenses the laser light on a specific part of the specimen without using a rotating disk, The second scanning optical system condenses the laser light on a specific part of the specimen without using a rotating disk,
    The focal planes of the first optical system and the second scanning optical system are at the same depth position, The focal planes of the first optical system and the second scanning optical system are at the same depth position,
    The microscope apparatus according to claim 2, wherein the second scanning optical system irradiates the specific portion with the laser beam by a scanning unit that arbitrarily deflects the laser beam in the XY directions. The microscope apparatus according to claim 2, wherein the second scanning optical system irradiates the specific portion with the laser beam by a scanning unit that appropriately deflects the laser beam in the XY directions.
  2. 請求項記載の顕微鏡装置において、前記第2の走査光学系が前記レーザ光源のレーザ光のビーム径を変化させるビーム径可変機構を更に備えることを特徴とする顕微鏡装置。 The microscope apparatus according to claim 1 , wherein the second scanning optical system further includes a beam diameter variable mechanism that changes a beam diameter of the laser light of the laser light source.
  3. コヒーレントではない光源から出力された光によって対物レンズを介して標本を走査し、前記標本からの蛍光を前記対物レンズを介して検出する第1の走査光学系と、
    レーザ光源から出力されたレーザ光を標本の特定の部位に照射し、特異現象を発現させるための第2の走査光学系とを具備し、 It is provided with a second scanning optical system for irradiating a specific part of the specimen with a laser beam output from a laser light source to cause a peculiar phenomenon to appear.
    前記第1の走査光学系は、共焦点効果を得るための回転ディスクを更に具備し、 The first scanning optical system further includes a rotating disk for obtaining a confocal effect.
    前記コヒーレントではない光源から出力された光は、前記回転ディスクを介して標本を走査し、 The light output from the non-coherent light source scans the sample through the rotating disc and
    前記蛍光は、前記回転ディスクを介して検出され、 The fluorescence was detected via the rotating disc and
    前記第2の走査光学系は回転ディスクを介さずに前記レーザ光を前記標本の特定部位に集光し、 The second scanning optical system concentrates the laser beam on a specific part of the sample without going through a rotating disk.
    前記第1の光学系及び前記第2の走査光学系の焦点面は同じ深さ位置にあり、前記第2の走査光学系が前記レーザ光源のレーザ光のビーム径を変化させるビーム径可変機構を更に備えることを特徴とする顕微鏡装置。 The focal planes of the first optical system and the second scanning optical system are at the same depth position, and the second scanning optical system provides a beam diameter variable mechanism for changing the beam diameter of the laser beam of the laser light source. A microscope device further comprising. A first scanning optical system that scans a sample through an objective lens with light output from a non-coherent light source, and detects fluorescence from the sample through the objective lens; A first scanning optical system that scans a sample through an objective lens with light output from a non-coherent light source, and detects fluorescence from the sample through the objective lens;
    A second scanning optical system for irradiating a specific portion of the specimen with a laser beam output from a laser light source and causing a specific phenomenon to occur, A second scanning optical system for irradiating a specific portion of the specimen with a laser beam output from a laser light source and causing a specific phenomenon to occur,
    The first scanning optical system further comprises a rotating disk for obtaining a confocal effect, The first scanning optical system further a rotating disk for obtaining a confocal effect,
    The light output from the non-coherent light source scans the specimen through the rotating disk, The light output from the non-coherent light source scans the specimen through the rotating disk,
    The fluorescence is detected via the rotating disk; The fluorescence is detected via the rotating disk;
    The second scanning optical system condenses the laser light on a specific part of the specimen without using a rotating disk, The second scanning optical system condenses the laser light on a specific part of the specimen without using a rotating disk,
    The focal planes of the first optical system and the second scanning optical system are at the same depth, and the second scanning optical system has a beam diameter variable mechanism that changes the beam diameter of the laser light of the laser light source. A microscope apparatus further comprising: The focal planes of the first optical system and the second scanning optical system are at the same depth, and the second scanning optical system has a beam diameter variable mechanism that changes the beam diameter of the laser light of the laser light source. further comprising: further comprising:
  4. コヒーレントではない光源から出力された光によって対物レンズを介して標本をケーラー照明し、前記標本からの蛍光を前記対物レンズを介して検出する第1の光学系と、
    レーザ光源から出力されたレーザ光を標本の特定の部位に集光して照射し、特異現象を発現させるための第2の走査光学系とを具備し、

    前記第1の光学系及び前記第2の走査光学系の焦点面は同じ深さ位置にあり、 The focal planes of the first optical system and the second scanning optical system are at the same depth position.
    前記第2の走査光学系が前記レーザ光源のレーザ光のビーム径を変化させるビーム径可変機構を更に備えることを特徴とする顕微鏡装置。 A microscope apparatus in which the second scanning optical system further includes a beam diameter variable mechanism for changing the beam diameter of the laser beam of the laser light source. A first optical system for Koehler illuminating the specimen via an objective lens with light output from a non-coherent light source, and detecting fluorescence from the specimen via the objective lens; A first optical system for Koehler illuminating the specimen via an objective lens with light output from a non-coherent light source, and detecting fluorescence from the specimen via the objective lens;
    A second scanning optical system for condensing and irradiating a specific portion of the specimen with the laser light output from the laser light source, and causing a specific phenomenon to occur, A second scanning optical system for condensing and irradiating a specific portion of the specimen with the laser light output from the laser light source, and causing a specific phenomenon to occur,
    The focal planes of the first optical system and the second scanning optical system are at the same depth position, The focal planes of the first optical system and the second scanning optical system are at the same depth position,
    The microscope apparatus, wherein the second scanning optical system further includes a beam diameter variable mechanism that changes a beam diameter of the laser light of the laser light source. The microscope apparatus, wherein the second scanning optical system further includes a beam diameter variable mechanism that changes a beam diameter of the laser light of the laser light source.
  5. 請求項 〜請求項記載の顕微鏡装置において、前記ビーム径可変機構より出力されるレーザ光のビーム径から標本面上での深さ方向の励起光強度分布を算出し、又は記憶する励起光強度分布算出手段を更に具備することを特徴とする顕微鏡装置。 In the microscope apparatus according to claim 2 to claim 4, wherein the beam-diameter to calculate the excitation light intensity distribution in the depth direction on the sample surface from the beam diameter of the laser beam output from the varying mechanism, or storage exciting light A microscope apparatus further comprising intensity distribution calculating means.
  6. 共焦点顕微鏡装置を用いた観察方法であって、
    回転ディスクを介して標本に励起光を照射し、ディスク走査により標本の蛍光像を取得する第1のステップと、 The first step of irradiating the sample with excitation light via a rotating disk and acquiring a fluorescence image of the sample by scanning the disk,
    特異現象を発現させるためのレーザ光を前記回転ディスクを介さずに前記標本の所望の位置に集光して照射する第2のステップとを具備し、 It comprises a second step of concentrating and irradiating a laser beam for expressing a peculiar phenomenon at a desired position of the sample without going through the rotating disk.
    前記第1及び第2のステップにおいて同じ深さ位置にある焦点面にそれぞれの光が照射され、 In the first and second steps, the focal planes at the same depth are irradiated with light, respectively.
    前記第2のステップは、前記標本の所望の位置に前記レーザ光を照射するようにそのレーザ光をXY方向に任意に偏向する走査手段によって偏向させるステップを含むことを特徴とする観察方法。 The second step is an observation method comprising a step of deflecting the laser beam by a scanning means that arbitrarily deflects the laser beam in the XY direction so as to irradiate the desired position of the sample with the laser beam. An observation method using a confocal microscope apparatus, An observation method using a confocal microscope apparatus,
    A first step of irradiating the specimen with excitation light via a rotating disk and acquiring a fluorescent image of the specimen by scanning the disk; A first step of irradiating the specimen with excitation light via a rotating disk and acquiring a fluorescent image of the specimen by scanning the disk;
    A second step of condensing and irradiating a laser beam for expressing a singular phenomenon at a desired position of the specimen without passing through the rotating disk, A second step of condensing and irradiating a laser beam for expressing a singular phenomenon at a desired position of the specimen without passing through the rotating disk,
    In the first step and the second step, the respective focal planes at the same depth position are irradiated with each light, In the first step and the second step, the respective focal planes at the same depth position are irregular with each light,
    The second step includes a step of deflecting the laser beam by a scanning unit that arbitrarily deflects the laser beam in the XY directions so that the laser beam is irradiated to a desired position of the specimen. The second step includes a step of irradiating the laser beam by a scanning unit that appropriately deflects the laser beam in the XY directions so that the laser beam is selectively to a desired position of the specimen.
  7. 請求項記載の観察方法において、前記第1又は第2のステップは、前記特異現象を発現させるための光の焦点面を同じ位置に保ったまま、その標本面における深さ方向の強度分布を調整するステップを更に含むことを特徴とする観察方法。 7. The observation method according to claim 6 , wherein the first or second step calculates an intensity distribution in a depth direction on the sample surface while maintaining a focal plane of light for expressing the singular phenomenon at the same position. An observation method, further comprising the step of adjusting.
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